Trafficking regulation of the chemokine receptor CCR2B compared to CCR5

The closely-related CC chemokine receptors 2B and 5 are seven-transmembrane domain receptors coupled to heterotrimeric G proteins. The two receptors bind inflammatory chemokines and play important complementary roles in the recruitment of specific leukocyte sub-populations to sites of infection. To enable fine-tuning of cellular responses to chemokines, CCR2B and CCR5, like other GPCRs, can be desensitised in response to agonist stimulation or cross-talk with other receptors. This involves down-modulation of cell surface active receptor through two essential transportation events, endocytosis and recycling. The CCR5 endocytic and recycling pathways are well established and several mechanisms involved have been clearly defined. Conversely, less is known about the route followed by CCR2B upon stimulation.

This study investigated the regulation, trafficking and fate of CCR2B in the context of THP-1 cells endogenously expressing the receptor and HEK293 transfectants. Comparison with CCR5 highlighted marked differences in the behavious of the two receptors. However, my initial findings indicate that certain aspects of the regulation of CCR5 as well as CCR2B may be cell type-dependent.

Flow cytometry, immunofluorescence and biochemical analyses showed that unlike CCR5, internalised CCR2B can be both degraded and recycled following agonist stimulation. In HEK293, CCR2B follows an EGF receptor-like pathway, transiting through early endosomes containing EEA1, transferrin and Rab4, reaching CD63 and Lamp1 positive late endosomes/lysosomes before being degraded.

Importantly, I showed that CCR2B cell surface molecules are N- and O-glycosylated, and only this glycosylated form of the receptor is targeted for agonist-induced degradation.

This thesis also presents findings from proteomics approaches developed in an attempt to identify interacting proteins implicated in the trafficking of each receptor.

This study brings new insights to the endocytic regulation of agonist-treated CC chemokine receptors, revealing receptor- and cell type-specific behaviours, which add complexity to a relatively conserved process.